Detecting Cells Secreting A Protein of Interest

ABSTRACT

In some cases, the described systems and methods include obtaining a cell sample containing multiple antibody-producing cells. In such cases, the cells can be tagged with a cross-linking reagent having a first portion configured to bind to a marker on the antibody-producing cells and a second portion configured to bind to an antigen of interest. In some instances, the tagged antibody-producing cells are exposed to the antigen of interest such that the antigen becomes bound to the cells. In some such instances, the antibody-producing cells are also allowed to produce an antibody, such that a portion of the antibody-producing cells produce an antigen-specific antibody that binds to the antigen of interest. To identify cells that produce the antigen-specific antibody, the tagged cells can be exposed to a labeled secondary antibody that is configured to bind to the antigen-specific antibody. Other implementations are also described.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a division of U.S. patent application Ser. No.14/044,559, filed Oct. 2, 2013 (pending), which is a non-provisional(and thus claims the benefit of the filing date) of U.S. provisionalpatent application Ser. No. 61/720,349 (filed Oct. 30, 2012). Both ofthe foregoing applications are incorporated by reference herein in theirentireties.

BACKGROUND

Antibodies, which are also known as immunoglobulins, are proteins thatare produced by B-cells and that are used by organisms' immune systemsto identify and neutralize foreign objects, such as bacteria, viruses,and other antigens. Generally, antibodies include a Y-shaped proteinhaving two large heavy chains and two small light chains. Additionally,while the general structure of antibodies is generally similar, a smallregion at the tip of such proteins can be extremely variable, allowingfor millions of antibodies with slightly different tip structures, orantigen binding sites. The region of a Y-shaped antibody protein thatincludes the antigen binding site is sometimes called the F_(AB) (or thefragment, antigen binding) region. This F_(AB) region, in turn, includesa constant domain (which remains constant across multiple types ofantibodies) and one or more variable or hypervariable regions (whichvary from one antibody to another) from each heavy and light chain ofthe antibody. The variable domains from the heavy chain (“V_(H)”) andlight chain (“V_(L)”), which are part of the antibody's variable-region(or V-region), may be the most important parts of an antibody, and areresponsible for binding the antibody to specific antigens.

While antibodies are well known for playing an important role inorganisms' natural immune responses, antibodies have proven useful for awide variety of other purposes. Indeed, antibodies have been found to beuseful in many forms of medical diagnosis (e.g., in pregnancy tests,lupus diagnostic tests, etc.), in many research applications (e.g., toidentify and locate intracellular and extracellular proteins), and evento treat a wide variety of diseases (e.g., rheumatoid arthritis,multiple sclerosis, psoriasis, cancer, etc.).

In the quest to find antibodies that can be used as therapeutictreatments for disease, there is a need to identify specific antibodiesthat are capable of effectively identifying and/or neutralizing antigensassociated with a wide variety of diseases and maladies. Additionally,as many conventional methods for identifying such antibodies can berelatively inefficient and ineffective at identifying effectiveantibodies, it would be an improvement in the art to augment or evenreplace current techniques with other techniques.

SUMMARY

This disclosure relates to systems and methods for detecting cells thatproduce a protein of interest, such as an antigen-specific antibody.While the described methods can involve any suitable element, in somenon-limiting implementations, the described methods include obtaining acell sample containing protein-producing cells. In some cases, theprotein-producing cells are then tagged with a cross-linking reagent,wherein the cross-linking reagent has a first portion that is adapted tobind to a cell surface marker that is specific to the protein-producingcells and a second portion that is configured to be bound to either anantigen of interest or a protein-specific antibody, which is an antibodythat is specific to the protein of interest. In some such cases, thedescribed methods further include exposing the protein-producing cellsto the antigen of the interest or the protein-specific antibody, suchthat the protein-producing cells become linked through the cross-linkingreagent to the antigen of interest or the protein-specific antibody. Thedescribed methods can further include allowing the protein-producingcells to produce the protein of interest, wherein the protein ofinterest is adapted to selectively bind with the antigen of interest orthe protein-specific antibody. In some cases, the methods furtherinclude identifying cells that produce the protein of interest byexposing the protein-producing cells to a labeled antibody that isadapted to bind to the protein of interest.

In some other non-limiting implementations, the described methodsinclude obtaining a cell sample containing multiple antibody-producingcells. In such cases, the antibody-producing cells can be tagged with across-linking reagent that has a first portion that is configured tobind to a marker that is specific to antibody-producing cells, and asecond portion that is configured to bind to an antigen of interest. Insome instances, the tagged antibody-producing cells are exposed to theantigen of interest such that the antigen becomes bound to the cells viathe cross-linking reagent. The antibody-producing cells are also allowedto produce antibodies, such that a portion of the antibody-producingcells produce an antigen-specific antibody that binds to the antigen ofinterest. To identify cells that produce the antigen-specific antibody,the tagged cells can be exposed to a labeled secondary antibody that isconfigured to bind to the antigen-specific antibody.

In still other non-limiting implementations, the described methodsinclude obtaining a cell sample containing protein-producing cells. Insome cases, the protein-producing cells are tagged with a cross-linkingreagent, wherein the cross-linking reagent has a first portion adaptedto bind to a cell surface marker that is specific to theprotein-producing cells and a second portion that is configured to bebound to an a protein-specific antibody, or an antibody that is specificto the protein of interest. In some cases, the described methods furtherinclude exposing the protein-producing cells to the protein-specificantibody, such that the protein-producing cells become linked throughthe cross-linking reagent to the protein-specific antibody. In some suchcases, the protein-producing cells are also allowed to produce theprotein of interest. Additionally, to identify cells that produce theprotein of interest, the protein-producing cells are exposed to alabeled antibody that is adapted to bind to the protein of interest.

These and other features and advantages of the described systems andmethods will be set forth or will become more fully apparent in thedescription that follows and in the appended claims. The features andadvantages may be realized and obtained by means of the instruments andcombinations particularly pointed out in the appended claims.Furthermore, the features and advantages of the described systems andmethods may be learned by their practice or will be obvious from thedescription, as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flowchart depicting some embodiments of a method fordetecting cells that produce a protein of interest;

FIGS. 2A-2B each illustrate some embodiments of a cross-linking reagent;

FIGS. 3A-3G depict some embodiments of the method for detecting cellsthat produce the protein of interest;

FIGS. 4A-4G depict some alternative embodiments of the method fordetecting cells that produce the protein of interest;

FIGS. 5A-5F depict some alternative embodiments of the method fordetecting cells that produce the protein of interest;

FIG. 6 shows a flowchart depicting some alternative embodiments of themethod for detecting cells that produce the protein of interest; and

FIGS. 7A-7F depict some additional embodiments of the method fordetecting cells that produce the protein of interest.

The Figures illustrate specific aspects of the described systems andmethods for detecting cells secreting a protein of interest. Togetherwith the following description, the Figures demonstrate and explain theprinciples of the structures, methods, and principles described herein.In the drawings, the thickness, size, and proportion of elements may beexaggerated or otherwise modified for clarity. Moreover, for clarity,the Figures may show simplified or partial views of the describedmethods and their associated components and reagents.

DETAILED DESCRIPTION

The following description supplies specific details in order to providea thorough understanding. That said, to avoid obscuring aspects of thedescribed systems and methods for detecting cells secreting a protein ofinterest, well-known structures, materials, processes, techniques, andoperations are not shown or described in detail. Additionally, theskilled artisan will understand that the described systems and methodscan be implemented and used without employing these specific details.Indeed, the described systems and methods can be placed into practice bymodifying the illustrated methods and reagents and can be used inconjunction with any other apparatus and techniques conventionally usedin the industry. For example, while the description below focuses onmethods that include the use of cross-linking reagents, labeledsecondary antibodies, labeled antibodies, beads and/or cells having anantigen (or antibody or other protein) of interest on their surface, andother devices and process, the described systems and methods (orportions thereof) can be used with any other suitable reagents, devices,or techniques. For instance, instead of (or in addition to) using thedescribed systems and methods to detect cells that produce anantigen-specific antibody, the described systems and methods can bemodified to allow for the detection of cells that produce any othersuitable protein of interest.

As mentioned above, some embodiments of the described systems andmethods relate to systems and methods for detecting cells that produce aprotein of interest, such as an antigen-specific antibody. While thedescribed methods can involve any suitable element, in some cases, theyinclude obtaining a cell sample containing multiple antibody-producingcells. In such cases, the antibody-producing cells can be tagged with across-linking reagent that has a first portion that is configured tobind to a marker that is specific to antibody-producing cells, and asecond portion that is configured to bind to an antigen of interest. Insome instances, the tagged antibody-producing cells are exposed to theantigen of interest such that the antigen becomes bound to the cells viathe cross-linking reagent. In some such instances, theantibody-producing cells are also allowed to produce an antibody, suchthat a portion of the antibody-producing cells produce anantigen-specific antibody that binds to the antigen of interest. Toidentify cells that produce the antigen-specific antibody, the taggedcells can be exposed to a labeled secondary antibody that is configuredto bind to the antigen-specific antibody.

The described systems and methods thus generally provide techniques fordetecting cells that produce a protein of interest, such as an antibodythat binds to an antigen of interest (or an antigen-specific antibody).In this manner, the described systems and methods can be used toidentify and/or separate cells that produce a protein (e.g., antibody)having a desired characteristic from cells that do not produce theprotein of interest. Accordingly, some embodiments of the describedsystems and methods can be used to identify cells that produce anantigen-specific antibody that can be used in therapeutic treatments, indisease diagnosis, and/or for any other suitable application.

While the described methods can be used and accomplished in any suitablemanner, FIGS. 1 and 3A through 7F illustrate some embodiments ofsuitable methods for detecting cells that secrete a protein of interest(e.g., an antigen-specific antibody and/or another desired protein). Inthis regard, FIGS. 1 and 6 provide general overviews of some embodimentsof the described methods, and FIGS. 3A through 5F and 7A through 7Fprovide illustrations of some more-specific embodiments of the describedmethods. To provide a better understanding of the described methods, themethod 100 of FIG. 1 is first described, followed by a description ofthe methods in FIGS. 3A through 7F.

Each of the methods described herein can be modified in any suitablemanner (including by rearranging, adding to, removing, substituting, andotherwise modifying various portions of the methods). Moreover, in someembodiments, at any suitable place in the methods, cells can beselected, moved, sorted, and/or the like through the use ofopto-electrical techniques, such as opto-electrical wetting (“OEW”) asdisclosed, for example, in U.S. Pat. No. 6,958,132; opto-electronictweezers (“OET”) as disclosed, for example, in U.S. Pat. No. 7,612,355;cell shooting; and/or any other suitable technique that can be used toselect, move, sort, filter, or otherwise manipulate cells. In thisregard, an example of a cell shooter device is disclosed in U.S.Provisional Patent Application Ser. No. 61/653,322, filed May 30, 2012and having an attorney docket no. BL1-PRV (hereinafter the “'322Application”), which is incorporated by reference herein in itsentirety. Indeed, the sorting, selecting, moving, and/or the like of thecells can be performed, for example, in the processing/outputting device100 of the '322 Application in the same way that the cells 120 aresorted, selected, moved, and/or the like in the device 100 as shown inthe Figures of the '322 Application. Moreover, one or more of the cellscan then be expressed in a droplet of medium as a cell 120 is expressedin a droplet 706 as illustrated in and discussed with respect to FIGS.7A-11 of the '322 Application. Accordingly, in some embodiments, thecells can thus be transferred during one or more of the described stepsof the instant application from one device to another, generally asillustrated in FIGS. 12-15 of the '322 Application.

Now, with reference to FIG. 1, that Figure shows (at 105) that someembodiments of the method 100 begin by obtaining a sample ofprotein-producing cells. In this regard, the term protein-producing cellmay refer to any cell that is capable of secreting, displaying on itsmembrane, or otherwise producing a protein, such as a hormone, enzyme,functional protein, structural protein, and/or antibody. For the sake ofsimplicity, however, the method 100 of FIG. 1 focuses onprotein-producing cells that produce an antibody (or antibody-producingcells). Some examples of such antibody-producing cells include, but arenot limited to, B-lymphocytes, plasma cells, plasmablasts, hybridomas,activated B cells, memory cells, transformed cells, mammalian cells thatexpress immunoglobulin genes or parts thereof, and/or any other cellsthat are able to produce an antibody.

While the sample of antibody-producing cells can be collected in anysuitable manner and from any suitable location, in some embodiments,cells of a B-Cell lineage are surgically extracted (or otherwiseremoved) from an animal's spleen, lymphatic tissue, bone marrow, blood,and/or other suitable tissue or fluid. In this regard, any suitablenumber of cells can be collected.

With continued reference to FIG. 1 (at 105), in some embodiments, atleast some of the cells in the sample of antibody-producing cells havebeen involved in an immune response. While this immune response can betriggered in any suitable manner, in some embodiments, an animal (e.g.,a human, rabbit, rat, mouse, goat, cow, chimpanzee, monkey, or otherorganism); tissue; or cell population is exposed to an antigen ofinterest (e.g., via immunization, pathogen infection, pathogenesis, anauto-immune reaction, and/or a similar method) or is already sufferingfrom a disease or pathological condition of interest. In this regard,and as used throughout this specification, the term antigen may refer toany known or unknown substance that can be recognized by an antibody,including, without limitation, any suitable protein, glycoprotein,and/or carbohydrate. In one example, an antigen is a biologically-activeprotein, such as one or more hormones, cytokines, and their cellreceptors; bacterial or parasitic cell membranes or purified componentsthereof; and/or viral components. Additionally, while antigens can be inany suitable form, in some cases, antigens are available in a pure formobtained either by direct purification from the native source or byrecombinant expression and purification of such antigens. In othercases, antigens are expressed on the surface of a cell, either naturallyor recombinantly. In such cases, antigen expression can occur on, but isnot limited to, mammalian cells, immunomodulatory cells, lymphocytes,monocytes, polymorphs, T cells, tumor cells, yeast cells, bacterialcells, infectious agents, parasites, plant cells, transfected cells(e.g., NSO, CHO, COS, 293 cells, etc.), cells associated with aparticular disease or malady, and/or any other suitable cell orpopulation of cells.

Continuing with the method 100, FIG. 1 (at 110) shows that, in someembodiments, the method 100 includes the preparation (or acquisition) ofone or more cross-linking reagents. Such cross-linking reagents cancomprise any suitable component and can be prepared in any suitablemanner that allows a first portion of the cross-linking reagent to bindto a cell surface marker on an antibody-producing cell and a secondportion of the reagent to bind to an antigen of interest. By way ofillustration, FIGS. 2A and 2B show some embodiments in which thecross-linking reagent 10 comprises a first portion 15, a cross-linker20, and second portion 25.

With respect to the first portion 15 of the cross-linking reagent 10,the first portion can comprise any suitable component that allows it tobind to a cell surface marker that is specific to a desired cell type.In this regard, the term cell surface marker may be used to refer to anyprotein expressed on a surface of a protein-producing cell (e.g., anantibody-producing cell) that is capable of being used to differentiatea cell expressing that protein from another cell that does not expressthe same protein. For instance, some examples of plasma cell surfacemarkers include, but are not limited to, CD138, CD38, CD78, and theinterleukin-6 receptor.

While the first portion 15 of the cross-linking reagent 10 can be madein any suitable manner, in some embodiments, an antibody that isspecific to a cell surface marker (e.g., CD138) that is expressed on thesurface of a desired protein-producing cells (e.g., orantibody-producing cell, such as a plasma cell) is obtained. In someembodiments, a F_(AB) fragment (which may be used herein to refer toF_(AB) fragments, F_(AB)' fragments, and/or F(_(AB)')_(x) fragments) ofthat antibody is then prepared and purified. In this regard, the F_(AB)fragment of the antibody can be prepared in any suitable manner,including by using one or more enzymes (e.g., papain, pepsin, animmunoglobulin degrading enzyme from Streptococcus pyogenes, aproteolytic enzyme, etc.) to cleave the antibody and produce one or moreF_(AB) fragments. In some optional embodiments, to make the firstportion of the cross-linking reagent, the variable regions of the heavyand light chains of the antibody are fused together to form asingle-chain variable fragment (“scFv”) that is about half the size ofits corresponding F_(AB) fragment, and that retains the originalspecificity of the parent antibody.

With regards to the cross-linker 20, the cross-linker can comprise anysuitable chemical and/or biological linking agent having at least tworeactive end groups that react with functional groups (e.g., primaryamines, carboxyls, carbonyls, and/or sulfhydryls). Some examples of suchlinking agents include, but are not limited to, one or morehetero-bifunctional reagents, homo-bifunctional reagents, avidin-biotincross-linkers, and streptavidin-biotin cross-linkers. According to someembodiments, however, FIG. 2B shows the cross-linker 20 comprises astreptavidin-biotin cross-linker 30 (wherein SA stands for streptavidinand B stands for biotin).

Turning now to the second portion 25 of the cross-linking reagent 10,the second portion can comprise any suitable component that allows it tobind to an antigen of interest and/or to a cell or bead bearing anantigen (or antibody or other protein) of interest on the cell/bead'souter surface. Some examples of suitable components that can be used asthe second portion of the cross-linking reagent include, withoutlimitation, a F_(AB) fragment that is specific to the antigen ofinterest (e.g., a F_(AB) made from an antigen-specific antibody), aF_(AB) fragment that is specific to another protein of interest (e.g.,an antibody or other protein), an scFv that is specific to the antigenof interest (e.g., an scFv made from an antigen-specific antibody), aF_(AB) fragment which binds to a cell surface marker that is specific tothe cell expressing the antigen of interest on the cell's membranesurface, a cross-linker (e.g., cross-linker 30) that is capable ofbinding to the antigen of interest (e.g., the antigen (or Ag) 40 shownin FIG. 2B), and/or a cross-linker that is capable of binding to a celland/or bead bearing a protein (e.g., an antibody) of interest. By way ofillustration, FIG. 2A shows some embodiments in which the second portion25 of the cross-linking reagent 10 comprises a F_(AB) fragment that isspecific to the antigen of interest. In contrast, FIG. 2B shows that, insome embodiments, the second portion 25 of the cross-linking reagent 10and the cross-linker 20 are one and the same. Thus, FIG. 2B shows thatthe cross-linker 20 itself can act as the second portion 25 and be boundto the antigen of interest 40.

The cross-linking reagent 10 can be made in any suitable manner,including, without limitation, through derivitization of proteins (e.g.,amine derivitization, disulfide formation, carboxylic acidderivitization, etc.), activated-ester-based coupling, biotinylation,and/or any other suitable method. In some embodiments, however, thefirst portion 15 (e.g., the first F_(AB) fragment) and the secondportion 25 (e.g., the second F_(AB) fragment) of the cross-linkingreagent are biotinylated, mixed at approximately a 1:1 ratio, andcross-linked with streptavidin. In such embodiments, a tetramer with astatistical mixture of the first and second portions (e.g., F_(AB)fragments) is made.

Continuing with the method 100 and returning to FIG. 1, that Figureshows (at 115) that some embodiments of the method include washing thesample of antibody-producing cells to remove antibody that is free insolution. In this regard, the cells can be washed in any suitablemanner, including, without limitation, by being rinsed and/or beingplaced in one or more baths (e.g., water baths, cell-support mediabaths, etc.), through filtration (e.g., light filtration, microfluidicfiltration, etc.), OET, OEW, and/or any other suitable method that iscapable of retaining the antibody-producing cells while allowingfree-floating antibodies to be washed away.

At 120, FIG. 1 shows the antibody-producing cells are treated with anysuitable amount (e.g., an excess) of the cross-linking reagent 10 thatallows such cells to be tagged with that reagent. In this manner, thefirst portion 15 of the cross-linking reagent is able to bind to thecell surface markers (e.g., CD138) of antibody-producing cells, whileleaving cells that lack the desired cell surface markers (e.g.,non-antibody-producing cells) substantially untagged.

At 125, FIG. 1 shows that some embodiments of the method 100 continue asthe sample of cells (including the antibody-producing cells that aretagged with the cross-linking reagent 10) is treated with the antigen ofinterest 40. While this can be done in any suitable manner, in someembodiments, an antigen of interest is bound to (or expressed on) thesurface of cells that are exposed in excess to the taggedantibody-producing cells. In other embodiments, an antigen of interestis bound to the surface of beads (e.g., via a chromic-chloride couplingmethod, water-soluble-carbodiimide-coupling method, or other suitablemethod) and exposed, in excess, to the tagged antibody-producing cells.In still other embodiments, a free form of the antigen of interest(e.g., a form in which the antigen of interest is not bound to cells orbeads, such as a purified form of the antigen) is added to the taggedantibody-producing cells. In yet other embodiments, the antigen ofinterest is already bound to the cross-linking reagent 10 (as shown inFIG. 2B) when the antibody-producing cells are tagged with the reagent.In any case, as a result of treating the tagged antibody-producing cellswith an excess of the antigen of interest, the antibody-producing cellsare bound through the cross-linking reagent to the antigen of interest(e.g., via antigen-coated cells, antigen-coated beads, free-floatingantigens, and/or a cross-linker comprising the antigen of interest).

At 130, FIG. 1 shows that, in some embodiments, the excess antigen ofinterest (or the antigen that is not bound to the taggedantibody-producing cells via the cross-linking reagent 10 is removed. Inthis regard, the excess antigen (e.g., the excess antigen-coated cells,antigen-coated beads, free-floating antigen, and/or a cross-linkingreagent comprising the antigen of interest) can be removed in anysuitable manner, including, without limitation, through filtration withOET, light filtration, and/or another microfluidic filtration technique.In one example, OET is used to create a light comb with a tine having awidth that is configured to retain large materials (e.g.,antibody-producing cells complexed through the cross-linking reagent 10with the cells/beads displaying the antigen of interest, free floatingantigen, etc.) while allowing small materials (e.g., excessantigen-coated cells/beads, free-floating antigens, cross-linkingreagents, etc.) to be carried away by a flow of liquid.

At 135, FIG. 1 shows that, in some embodiments, the taggedantibody-producing cells that have been linked through the cross-linkingreagent 10 to the antigen of interest (e.g., to antigen-coated cells,antigen-coated beads, free-floating antigen, and/or a cross-linkingreagent comprising the antigen of interest) are incubated to allow thecells to produce antibodies. In this regard, antibodies that arespecific to the antigen of interest (or antigen-specific antibodies)will bind preferentially to the antigen of interest (e.g., to theantigen that is bound to the antibody-producing cells through thecross-linking reagent and beads comprising the antigen, cells comprisingthe antigen, free-floating antigen, and/or antigens that are included inthe cross-linking reagent). More specifically, the antigen-specificantibody will bind preferentially to the antigen surrounding theantibody-producing cells that secreted that antibody.

FIG. 1, at 140, shows that some embodiments of the method 100 continueas the antigen-specific antibodies that are bound to the antigen ofinterest are marked or otherwise labeled. In this regard, the termlabel, and variations thereof, may refer to any suitable probe, marker,or label that can be connected to an antibody and be used to distinguishstructures that are bound to the label from those that are not. In thisregard, some examples of suitable labels include, but are not limitedto, luminescent labels, radiolabels, and fluorescent labels (e.g., Aqua,Texas-Red, FITC, rhodamine, rhodamine derivative, fluorescein,fluorescein derivatives, cascade blue, Cy5, phyocerythrin, etc.).

While the labeled secondary antibody can be used in any suitable manner,in some embodiments, the labeled secondary antibody is added, in excess,to the solution containing tagged cells that are linked to the antigenof interest (e.g., via the cross-linking reagent 10). Additionally,although the labeled secondary antibody can have specificity for any orall Antibodies—either in their entirety or for portions thereof (e.g.,the F_(C) region, a F_(AB) region, etc.), in some embodiments, thelabeled secondary antibody is specific to the F_(C) portion of the heavychain of antibodies. As a result, in such embodiments, the labeledsecondary antibody can bind to any antibodies in the sample (includingantigen-specific antibodies that are bound to the antigen of interest,as well as to those antibodies that do not bind to the antigen ofinterest). Thus, cells that produce the antigen-specific antibody willbe bound to the antigen of interest (e.g., via the cross-linking reagent10), which in turn is bound to the antigen-specific antibody, and whichin turn is bound to the labeled secondary antibody.

Continuing with FIG. 1, that Figure shows (at 145) that in someembodiments, once the labeled secondary antibody has been bound with theantigen-specific antibody (and/or any other antibodies), the excesslabeled secondary antibody and/or non-antigen-specific antibodies areoptionally washed away. In this regard, the excess labeled secondaryantibody that is not bound to the antibody-producing cells (e.g., viathe antigen-specific antibody, which is bound to the antigen ofinterest, and which is bound to the antibody-producing cell through thecross-linking reagent 10) is optionally washed away, or otherwiseseparated from, the antibody-producing cells in any suitable manner. Asa result, some, if not all, of the potential background noise will alsobe washed away.

At 150, FIG. 1 shows that, in some embodiments, the antibody-producingcells that produce the antigen-specific antibodies (and which are,therefore, labeled with the labeled secondary antibody) are separatedfrom the remaining cells that do not produce the antigen-specificantibody (and which are not labeled to the same extent, if at all). Inthis regard, the cells producing the antigen-specific antibody can beseparated from the remaining cells in any suitable manner that allowscells producing the antigen-specific antibody (or labeled cells) to beidentified and be separated from the cells that are not labeled (or thatare not labeled to the desired extent).

Some examples of suitable methods for separating the labeled cells fromnon-labeled cells include, but are not limited to, the use of OET, otherOET-based methods, OEW, micromanipulation, laser capture,micro-pipetting, flow cytometry, etc. Additionally, in some non-limitingembodiments, labeled cells are placed in holding pens, such as thevirtual holding pens 714, 716, 718, and 720 of the device 100illustrated in FIG. 7C and throughout other Figures of U.S. ProvisionalPatent Application Ser. No. 61/720,956, filed Oct. 31, 2012 and havingan attorney docket no. BL6-PRV (hereinafter the “'956 Application”),which is incorporated by reference herein in its entirety. Thereafter,process 1100 of FIG. 11 of the '956 Application can optionally beperformed to further identify and select the cells based on the cells'ability to produce the antibody of interest. As discussed in the '956Application, the pens 714, 716, 718, and 720 can be virtual pens orphysical pens. Moreover, if virtual pens, the virtual pens can becreated, moved, and/or manipulated and the cells can be selected, moved,and/or manipulated within the device 100 of the '956 Application, forexample, using OET techniques generally as illustrated in FIG. 2 of the'956 Application or similar such technologies.

Once the labeled cells are separated from the non-labeled cells, FIG. 1(at 155) shows that, in some embodiments, the antigen of interest can bereleased from those cells that have been identified as producing theantigen-specific antibody. Thus, where the antigen of interest is boundto beads or cells, such beads and/or cells can be released from thegathered cells that produce the antigen-specific antibody (or collectedcells). Similarly, where the antigen of interest is bound directly tothe cross-linker 20 (as shown in FIG. 2B), the antigen can be released.While the antigen of interest can be removed from the collected cells inany suitable manner, in some embodiments, the collected cells aretreated with a proteolytic enzyme (e.g., trypsin and/or any othersuitable proteolytic enzyme), heated, treated with chemicals that cleaveor remove the cross-linker 20, and/or otherwise treated to remove/cleaveany suitable portion of the cross-linking reagent and/or antigen.

Once the cells that produce the antigen-specific antibody have beenidentified and separated from other cells that do not produce theantigen-specific antibody (or desired amounts thereof), FIG. 1 at 160shows that the cells producing the antigen-specific antibody and/or thecorresponding antigen-specific antibodies themselves are optionallytested to determine whether the antigen-specific antibody has one ormore desired characteristics (e.g., biochemical activity,antigen-specificity, antibody-binding affinity, is produced at a desiredquantity, is produced at a desired concentration, etc.). In this regard,the collected cells and/or the antigen-specific antibody they producecan be tested in any suitable manner, including, without limitation,through one or more biochemical assays (e.g., to test the ability of acandidate antibody to augment or disrupt a biochemical pathway ofinterest), antibody-binding affinity assays, antigen-specificity assays,etc. Additionally, while the collected cells that produce theantigen-specific antibody can be tested collectively, in some preferredembodiments, such cells and/or the antibody they produce are tested onan individual cell basis.

In some embodiments, the collected cells that produce theantigen-specific antibody are each tested for one or more desiredcharacteristics in physical chambers. While these chambers can have anysuitable component or characteristic, in some embodiments, the chambersare the same as, or similar to any of the cell interrogation devices100, 100, 400, 800, and 800′ and the cell apparatus 1000 illustrated inthe Figures of and discussed in U.S. Provisional Patent Application Ser.No. 61/664,421, filed Jun. 26, 2012 and having an attorney docket no.BL3-PRV (hereinafter the “'421 Application”), which is herebyincorporated in its entirety. As discussed therein and with reference toFIGS. 2 and 3 of the '421 Application, such devices 100, 100′ caninclude a chamber 114 and/or 114′ for holding a cell 204 in a medium202. New medium 202 can be introduced through an inlet 110 and medium202 containing secretions from the cell 204 can be extracted through anoutlet 112 and analyzed.

Another example of the physical chambers into which a cell or cells canoptionally be placed for testing at step 160 of FIG. 1 of the instantapplication includes the cell secretion detection devices 100 and 100′illustrated in the Figures of and discussed in U.S. Provisional PatentApplication Ser. No. 61/709,408, filed Oct. 4, 2012 and having anattorney docket no. BL5-PRV (hereinafter the “'408 Application”), whichis incorporated by reference herein in its entirety. As discuss thereinand with reference to FIGS. 1A-3 of the '408 Application, such devicescan include a chamber 120 and/or 120′ for holding a cell 128 in a medium130 and a sensor 140 comprising one or more binding regions 142-148. Asillustrated in FIGS. 5A-6 of the '408 Application, the medium 130 cancomprise radiant labels 502 that bind to analytes 508 of interestsecreted by the cell 128. By monitoring with a detector 160 (see FIG. 1Dof the '408 Application) radiation of the binding regions 142-148 as thesecreted analytes 508 bind to those regions 142-148, the secretion ofthe analytes 508 can be measured and/or analyzed.

As noted, the chambers into which the cells are optionally placed atstep 160 can be the same as or similar to the devices noted above fromthe '421 Application or the '408 Application. If the devices of the '421Application, step 160 of the instant application can be performed, atleast in part, by extracting through the outlet 112 medium 202 andanalyzing the secretions from the cell 204 in the extracted medium 202(see FIGS. 2 and 3 of the '421 Application) generally as discussed inthe '421 Application. If the devices of the '408 Application, step 160of the instant application can be performed, at least in part, bydetecting and analyzing radiation from the binding regions 142-148 ofthe sensor 140 as briefly discussed above and in accordance with themore detailed discussions in the '408 Application.

As another example, at step 160 of the FIG. 1 in the instantapplication, the labeled cells can be placed in cell interrogationdevices in which the cells are held in a medium, which can further betested for the presence of the desired antibody. Examples of such cellinterrogation devices include the cell interrogation devices 100, 100′,400, 800, and 800′ and the cell apparatus 1000 as illustrated in theFigures of and discussed in the '421 Application. Other examples of suchcell interrogation devices include the cell secretion detection devices100 and 100′ as illustrated in the Figures of and discussed in the '408Application. The foregoing cell interrogation devices of the '421Application and the '408 Application can be operated generally in anymanner discussed in the '421 Application or the '408 Application but todetect secretion by the cells of the antibody of interest.

At 165, FIG. 1 shows that, in some embodiments, after a desiredbiochemical activity and/or other characteristic (e.g., antibody-bindingaffinity, antigen-specificity, concentration, etc.) of theantigen-specific antibody produced by the collected cells has beenmeasured, a cell line is optionally created that includes a portion ofthe DNA sequence (e.g., a copy of antibody V-region DNA sequence) fromthe collected cells that produce the antigen-specific antibody (or otherprotein of interest) having one or more desired characteristics. In thisregard, such a cell line can be produced in any suitable manner. Indeed,in some embodiments a portion of the DNA sequence (e.g., a copy of theV-region DNA sequence) from the collected cells that produce theantigen-specific antibody (or other protein of interest) having one ormore desired characteristics is optionally placed into a plasmid (e.g.,a plasmid encoding an immunoglobulin constant region), which can betransfected into a cell line that will produce the desiredantigen-specific antibody (or other protein). In some embodiments, theentire antibody (or other protein) genes from one or more of thecollected cells are cloned and/or sequenced. In other preferredembodiments, the variable regions or portions thereof (e.g., the V_(L)and/or V_(H)), which confer the desired specificity of the antibody, arecloned and/or sequenced, and then synthesized through any conventionaltechnique to produce recombinant antibodies. In this regard, recombinantantibodies can take several forms, including, without limitation, intactimmunoglobulins, chimeric antibodies, humanized antibodies, antigenbinding fragments (e.g., Fc, F_(AB), F_(AB)', and F(_(AB)')_(x)fragments, and derivatives thereof, such as scFv fragments).

Where the variable region of the DNA from the selected cells is clonedinto a plasmid, the cloning process can be accomplished in any suitablemanner. In some embodiments, however, the cloning process includes,lysing one of the collected cells (e.g., a cell producing anantigen-specific antibody 75 that has one or more desiredcharacteristics), extracting mRNA from the lysed cell (e.g., via a beadconjugated with a poly(T) tail); creating cDNA from the mRNA through areverse transcription polymerase chain reaction using PCR primersspecific to amplify the V_(H) and V_(L) regions; cloning the V_(H) andV_(L) regions into a plasmid containing an immunoglobulin constantregion; and transfecting the plasmid into a cell line (e.g., e. coli,CHO, HEK, NSO, or other suitable eukaryotic or mammalian cell line) tomake a stable antibody-secreting line (or other protein-producing cellline).

Additionally, in some alternative embodiments, the primary sequence ofthe antibody (or other protein) produced by the selected cells issequenced. More specifically, in some embodiments, the V-region DNAsequence from selected cells is sequenced, synthesized, and cloned intoa plasmid. In this regard, the sequencing, synthesis, and cloningprocesses can be accomplished in any suitable manner, including, withoutlimitation, through techniques that are commonly known in the art.

In still other embodiments, cells that have been identified as producinga protein of interest (e.g., an antigen-specific antibody) can be grown(e.g., in cell culture media) and then be fused with any other suitabletype of cell (e.g., myeloma cells) to form one or more hybridomas. Insome such embodiments, the fusion of the cell that is identified asproducing a protein of interest with another cell is a deterministicfusion that allows for the creation of a cell line that produces theprotein (e.g., antibody) of interest.

U.S. Provisional Patent Application 61/671,499 file Jul. 13, 2012 andhaving an attorney docket BL4-PRV (hereinafter the “'499 Application”),which is incorporated herein by reference in its entirety, illustratesexamples of techniques and devices that can be used to combine theplasmid into which the portion of the DNA sequence (e.g., the V-regionDNA sequence) of the antibody-producing cell 210 was placed with a cellof the cell line to be generated. That is, the plasmid into which theDNA sequence of the cell 210 was placed can be the micro-object 1 120and the cell of a cell line to be generated can be the micro-object 2122 in the Figures of the '499 Application. In some embodiments, theplasmid into which the DNA sequence of the cell 210 was placed can thusbe combined with the cell of a cell line to be generated using anyprocess and/or device illustrated in the Figures of and discussed in the'499 Application for combining the micro-object 1 120 with themicro-object 2 122 to produce the combined micro-object 124. Moreover,in some embodiments, the cell line referenced at step 135 of FIG. 1 ofthe instant application can be generated generally in accordance withthe process 600 or the process 1100 of FIG. 6 or 11 of the '956Application.

With reference now to FIGS. 3A through 3G, those Figures show someembodiments of the described methods in which beads or cells 35displaying the antigen of interest 40 are used to detect cells thatproduce an antigen-specific antibody. While the method of FIGS. 3Athrough 3G can be modified in any suitable manner, FIG. 3A shows that,in some embodiments, the method 200 involves exposing a washed sample ofantibody-producing cells 45 to a cross-linking reagent 10 having a firstF_(AB) fragment 50 that is specific to a cell surface marker 55 specificto the antibody-producing cells 45 (e.g., CD138 in some embodiments inwhich the antibody-producing cells comprise plasma cells) and a secondF_(AB) fragment 60 that is specific to the antigen of interest 40.

FIG. 3B shows that the tagged cell 65 is then exposed to cells and/orbeads 35 that display the antigen of interest 40 on their surface.Accordingly, FIG. 3C shows that the antigen coated cells and/or beads 35become linked to the antibody-producing cells 45 (e.g., via thecross-linking reagent 10). As discussed above, excess cells/beads 35 arethen optionally washed away (e.g., to reduce potential background).

FIG. 3D shows that as the antibody-producing cells 45 are incubated andallowed to produce antibodies 70, the antigen-specific antibody 75 willbind to the antigen of interest 40 that is linked (e.g., via beads/cells35 and cross-linking reagents 10) to the antibody-producing cells 80that produce the antigen-specific antibody 75. In contrast, FIG. 3Eshows that as cells 85 that produce non-antigen-specific antibodies 90are incubated and allowed to produce the non-antigen-specific antibody90, such antibodies do not bind significantly to the antigen of interest40.

Thus, in some embodiments, when a labeled secondary antibody 95 is added(e.g., a fluorescent labeled anti F_(C) antibody, as shown in FIGS. 3Dand 3E), the secondary antibody is allowed to bind with the antibodies70 produced by the antibody-producing cells, and antibodies that are notbound to the antigen of interest are optionally washed away. As aresult, FIGS. 3F and 3G illustrate that that the cells 80 that producethe antigen-specific antibody 75 are labeled with the labeled secondaryantibody 95, such that the labeled antigen-specific, antibody-producingcells (shown in 3F) are easily distinguishable (e.g., via fluorescentmicroscopy and/or any other suitable method) from the cells 85 that donot produce the antigen-specific antibody (shown in 3G).

Turning now to FIGS. 4A through 4G, those Figures show some embodimentsof the described methods in which free-floating antigens 98 are used todetect cells that produce an antigen-specific antibody 75. While themethod of FIGS. 4A through 4G can be modified in any suitable manner,FIG. 4A shows that, in some embodiments, the method 300 involvesexposing a washed sample of antibody-producing cells 45 to across-linking reagent 10 having a first F_(AB) fragment 50 that isspecific to a cell surface marker 55 on the antibody-producing cells 45and a second F_(AB) fragment 60 that is specific to the antigen ofinterest 40 (e.g., the free-floating antigen 98).

FIG. 4B shows that the tagged cell 65 is then exposed to thefree-floating antigen 98. Accordingly, FIG. 4C shows that thefree-floating antigen 98 becomes linked to the antibody-producing cells45 (e.g., via the cross-linking reagent 10). As discussed above, oncethe free-floating antigen has been linked to the tagged cell, excessfree-floating antigen is optionally washed away.

FIG. 4D shows that as the antibody-producing cells 45 are incubated andallowed to produce antibodies 70, the antigen-specific antibody 75 willbind to the antigen of interest 40 (e.g., the free-floating antigen 98)that is linked (e.g., via the cross-linking reagents 10) to theantibody-producing cells 80 that produce the antigen-specific antibody75. In contrast, FIG. 4E shows that as cells 85 that producenon-antigen-specific antibodies 90 are incubated and allowed to produceantibody 70, such non-antigen-specific antibodies 90 do not bindsignificantly to the antigen of interest 40 (e.g., antigen 98).

In some embodiments, when a labeled secondary antibody 95 is added tothe antibody-producing cells 45 (e.g., a fluorescent labeled anti F_(C)antibody, as shown in FIGS. 4D and 4E), the secondary antibody isallowed to bind with the antibodies 70 produced by theantibody-producing cells, and antibodies (e.g., non-antigen-specificantibodies 90 and labeled secondary antibody 95 bound thereto) that arenot bound the antigen of interest 40 (e.g., antigen 98) are optionallywashed away. As a result, FIGS. 4F and 4G illustrate that that the cells80 that produce the antigen-specific antibody 75 are labeled with thelabeled secondary antibody 95, such that the labeled cells (as shown inFIG. 4F) are easily distinguishable (e.g., via fluorescent microscopyand/or any other suitable detection method) from the cells 85 thatproduce non-antigen-specific antibodies (as shown in FIG. 4G).

With regards now to FIGS. 5A through 5F, those Figures show someembodiments of the described methods in which a cross-linking reagent 10comprising the antigen of interest 40 is used to detect cells thatproduce an antigen-specific antibody 75. Although the method of FIGS. 5Athrough 5F can be modified in any suitable manner, FIG. 5A show that, insome embodiments, the method 400 involves exposing a washed sample ofantibody-producing cells 45 to a cross-linking reagent 10 having a firstF_(AB) fragment 50 that is specific to a cell surface marker 55 on theantibody-producing cells 45, with the antigen of interest 40 bound tothe first F_(AB) fragment 50 through the cross-linker 20.

FIG. 5B shows that once the antibody-producing cells 45 are tagged withthe cross-linking reagent 10, the tagged cells 65 are also linked to theantigen of interest 40. As mentioned earlier (e.g., at 130 in FIG. 1),excess cross-linking reagent 10 is then optionally washed away.

FIG. 5C shows that as the tagged antibody-producing cells 65 areincubated and allowed to produce antibodies 70, the antigen-specificantibody 75 will bind to the antigen of interest 40 that is linked(e.g., via the cross-linking reagents 10) to the antibody-producingcells 80 that produce the antigen-specific antibody 75. In contrast,FIG. 5D shows that as cells 85 that produce non-antigen-specificantibodies 90 are incubated and allowed to produce antibody 70, suchnon-antigen-specific antibodies 90 do not bind, in significant numbers,to the antigen of interest 40.

As a result of the labeling described above, when a labeled secondaryantibody 95 is added to the cells 45 (e.g., a fluorescent labeled antiF_(C) antibody, as shown in FIGS. 5C and 5D), the secondary antibody isallowed to bind with the antibodies 70 produced by theantibody-producing cells, and excess antibodies (e.g.,non-antigen-specific antibodies 90 and secondary antibody 95 boundthereto) are optionally washed away. Accordingly, FIGS. 5E and 5Fillustrate that the cells 80 that produce the antigen-specific antibody75 are labeled with the labeled secondary antibody 95, such that thelabeled cells (as shown in FIG. 5E) are distinguishable (e.g., viafluorescent microscopy and/or any other suitable detection method) fromthe cells 85 that produce the non-antigen-specific antibody (shown inFIG. 5F).

As previously mentioned, the described methods can be modified in anysuitable manner. In one example (and as previously mentioned above),instead of just being used to identify cells that produce anantigen-specific antibody 75, the described methods can be modified toidentify cells that produce any other suitable protein of interest.

While the described systems and methods can be modified in any suitablemanner that allows them to be used to identify any suitable protein ofinterest, FIG. 6 shows some embodiments of one such method 500. In thisregard, FIG. 6 shows that, in at least some implementations, the method500 begins by obtaining a sample containing protein-producing cells. Inthis regard, this sample can be obtained in any suitable manner (e.g.,surgically or otherwise) and from any suitable location (e.g., from ananimal's spleen, lymphatic tissue, bone marrow, blood, and/or any othersuitable tissue or fluid that may include cells that produce a proteinof interest). Furthermore, the sample can contain any suitable number ofcells, and the cells can optionally be washed (e.g., in a manner similarto those discussed above with respect to step 115 of FIG. 1 of theinstant application).

At 510, FIG. 6 shows the method 500 further includes producing/obtainingone or more cross-linking reagents 20. In this regard, the cross-linkingreagents can have any suitable characteristic that allows them to beused to identify a protein of interest (e.g., including, withoutlimitation, any suitable characteristic discussed above with respect tostep 110 in FIG. 1 of the instant application). Indeed, in someembodiments, the cross-linking reagent 10 comprises a first portion 15(e.g., a F_(AB) fragment) that is configured to bind to a cell surfacemarker on cells (e.g., a specific type of cell) that produce proteins(e.g., a protein of interest, a specific type of protein, etc.).Additionally, in some embodiments, the cross-linking reagent comprises asecond portion 25 (e.g., a cross linker 20, a F_(AB) fragment, etc.)that is configured to bind (e.g., directly or indirectly) to a cell,bead, and/or other substrate bearing an antibody specific to the proteinof interest (or a protein-specific antibody). In still otherembodiments, the second portion of the cross-linking reagent (e.g., thecross linker) is configured to bind directly to the antibody that isspecific to the protein of interest.

Continuing with FIG. 6, at 515 the Figure shows that some embodiments ofthe method 500 include treating the protein-producing cells with anysuitable amount (e.g., an excess) of the cross-linking reagent 10 thatallows such cells to be tagged with that reagent. In this manner, thefirst portion 15 of the cross-linking reagent is able to bind to thecell surface markers 55 of the protein-producing cells (e.g., a certaintype of protein-producing cells), while leaving cells that do notinclude the desired cell surface markers substantially untagged.

At 520, FIG. 6 shows that some embodiments of the method 500 furtherincludes allowing the cross-linking reagent 10 to cross react with oneor more cells, beads, and/or other substrates that bear an antibodyspecific to the protein of interest. While this binding can beaccomplished in any suitable manner (e.g., by binding the cross-linkingreagent to the cells/beads/substrates via a F_(AB) fragment at thesecond portion 25 of the cross-linking reagent, via the cross-linker 20itself, or in any other suitable manner), in some embodiments, thecross-linker is bound to the cells/beads/substrates bearing the antibodyspecific to the protein of interest through the use of abiotin/streptavidin link.

At 525, FIG. 6 shows that, in some embodiments, the taggedprotein-producing cells 65 are allowed (e.g., incubated) to produceproteins. As a result, cells that produce the protein of interest willproduce proteins that will bind with the antibody on thebeads/cells/substrates.

Although the cells that produce the protein of interest can beidentified in any suitable manner, FIG. 6 (at 530) shows that, in someembodiments, the method 500 includes treating the tagged cells 65 thatare bound to the cells/bead/substrates with a labeled antibody (e.g., anexcess amount) that is specific to the protein of interest and thatbinds to a different epitope than that of the antibody on thecells/bead/substrates. In this regard, the labeled antibody can compriseany suitable label (e.g., one or more luminescent labels, radiolabels,and/or fluorescent labels, such as Aqua, Texas-Red, FITC, rhodamine,rhodamine derivative, fluorescein, fluorescein derivatives, cascadeblue, Cy5, phyocerythrin, etc.). In some embodiments, however, thelabeled antibody is labeled with one or more fluorescent molecules.

At 535, FIG. 6 shows that some embodiments of the method 500 optionallyinclude the removal of excess labeled antibody. While this removal canbe accomplished in any suitable manner, in some embodiments, excesslabeled antibody is simply washed away (e.g., as discussed above withrespect to step 145 of FIG. 1 of the instant application).

Additionally, through techniques similar to those discussed above withrespect to FIG. 1's steps 150, 155, 160, and 165, respectively, FIG. 6'ssteps 240, 545, 550, and 555 show that, in some embodiments, labeledcells are separated from non-labeled cells, the cross-linking reagent 10is removed from the protein producing cells, the characteristics of theproteins of interest produced by individual cells are optionally tested,and a cell line producing the protein of interest is optionally created.

To provide a better understanding of the method 500 described in FIG. 6,FIGS. 7A-7F illustrate some embodiments of a method 600 for identifyingcells that produce a protein of interest. In particular, FIG. 7A showsthat, in some implementations, the method 600 includes exposingprotein-producing cells 47 to a cross-linking reagent 10 having a firstportion 15 with specificity for a cell surface marker 55 on cellssecreting a protein (e.g., proteins in general, a certain type ofprotein, the protein of interest, etc.) and a second portion 20 that isconfigured to react (e.g., via a biotin/streptavidin cross-linker 20) tobead/cells 38 bearing an antibody 77 that is specific to the protein ofinterest 99. Accordingly, FIG. 7B shows that the protein-producing cells47 can be tagged (via the cross-linking reagent 10) with cells/beads 38that bear an antibody 77 specific to the protein of interest 99.

As the protein-producing cells 47 are allowed to secrete proteins, FIG.7C shows that the cells 48 that produce the protein of interest 99 havea relatively high amount of the protein of interest 99 bind to theantibody 77 bound to the cells/beads 38, at least when compared to cells49 that do not produce the protein of interest (e.g., as shown in FIG.7D). In this regard, while the antibody 77 bound to cells 49 that do notproduce the protein of interest (e.g., as shown in FIG. 7D) may stillbind to the protein of interest 99 released from nearby cells, it isbelieved that antibodies 77 that are tethered to the cells 48 thatproduce the protein of interest will bind with a significantly greateramount of the protein of interest than will antibodies that are tetheredto cells 49 that do not produce the antigen of interest.

Thus, FIGS. 7E and 7F show that when a labeled antibody 96 that isspecific to the protein of interest 99 is added to the tagged cells 65,cells 48 that produce the protein of interest (e.g., as shown in FIG.7E) are better labeled than cells 49 that do not produce the protein ofinterest (e.g., as shown in FIG. 7F). As a result of this labeling, thecells that produce the protein of interest will be preferentiallylabeled and can be separated from other cells with less intense or nolabeling. In this regard, the separation can be accomplished in anysuitable manner, including, without limitation, through the use of OET,an optoelectronic based method, micromanipulation, microfiltration, etc.

As mentioned previously, the described methods for detecting cells thatproduce a protein of interest can have several features. By way ofexample, some embodiments of the described systems and methods allow forindividual cells that produce a protein of interest (e.g., anantigen-specific antibody) to be identified and separated from cellsthat do not produce the protein of interest. In another example, someembodiments of the described methods allow cells that produce a higheramount of a protein of interest to be distinguished (e.g., by theincreased intensity of their labeling) from cells that produce a lesseramount or none of the protein of interest.

In still another example, some embodiments of the described methodsallow for the identification of cells that secrete a protein ofinterest, as opposed to cells that express a protein of interest on amembrane surface. In this regard, some conventional methods foridentifying cells that express a protein of interest rely on thestaining of a cell surface protein of interest. Accordingly, some suchconventional methods select for proteins of interest that have beenmodified post translation in such a way that they are bound to thecellular membrane, rather than identifying proteins of interest that aresecreted (as can be accomplished through some embodiments describedherein).

In addition to any previously indicated modification, numerous othervariations and alternative arrangements may be devised by those skilledin the art without departing from the spirit and scope of thisdescription, and appended claims are intended to cover suchmodifications and arrangements. Thus, while the information has beendescribed above with particularity and detail in connection with what ispresently deemed to be the most practical and preferred aspects, it willbe apparent to those of ordinary skill in the art that numerousmodifications, including, but not limited to, form, function, manner ofoperation, and use may be made without departing from the principles andconcepts set forth herein. Also, as used herein, the examples andembodiments, in all respects, are meant to be illustrative only andshould not be construed to be limiting in any manner. Furthermore, wherereference is made herein to a list of elements (e.g., elements a, b, c),such reference is intended to include any one of the listed elements byitself, any combination of less than all of the listed elements, and/ora combination of all of the listed elements. Also, as used herein, theterms a, an, and one may each be interchangeable with the terms at leastone and one or more. It should also be noted, that while the term stepis used herein, that term may be used to simply draw attention todifferent portions of the described methods and is not meant todelineate a starting point or a stopping point for any portion of themethods, or to be limiting in any other way.

What is claimed:
 1. A method for detecting production of anantigen-specific antibody, the method comprising: tagging anantibody-producing cell with a cross-linking reagent, wherein thecross-linking reagent comprises a first portion adapted to bind to acell surface marker that is specific to the antibody-producing cell anda second portion that comprises a protein of interest, wherein theantigen-specific antibody is specific to the protein of interest;allowing the antibody-producing cell to produce antigen-specificantibody; and exposing the antibody-producing cell to a labeledsecondary antibody configured to specifically bind to theantigen-specific antibody; and detecting whether the antibody-producingcell is bound to the labeled secondary antibody.
 2. The method of claim1, wherein the antibody-producing cell is an isolated antibody-producingcell.
 3. The method of claim 1, wherein the antibody-producing cell isone of multiple antibody-producing cells in a sample.
 4. The method ofclaim 1, wherein the first portion of the cross-linking reagentcomprises a F_(AB) fragment configured to bind to the cell surfacemarker.
 5. The method of claim 1, wherein the first portion of thecross-linking reagent comprises a ligand configured to bind to the cellsurface marker, and wherein the ligand does not comprise a F_(AB)fragment.
 6. The method of claim 1, wherein exposing the protein ofinterest is bound to a substrate.
 7. The method of claim 6, wherein theprotein of interest is bound to the substrate via a biotin/streptavidinlinkage.
 8. The method of claim 6, wherein the substrate is a bead, acell, or a combination thereof.
 9. The method of claim 1, wherein theprotein of interest is an antibody.
 10. The method of claim 1, whereinthe labeled secondary antibody is an anti-IgG antibody.
 11. A method fordetecting production of protein of interest, the method comprising:tagging a protein-producing cell with a cross-linking reagent, whereinthe cross-linking reagent comprises a first portion adapted to bind to acell surface marker that is specific to the antibody-producing cell anda second portion that comprises a first ligand, wherein the first ligandspecifically binds to the protein of interest; allowing theprotein-producing cell to produce the protein of interest; and exposingthe protein-producing cell to a labeled antibody configured tospecifically bind to the protein of interest; and detecting whether theprotein-producing cell is bound to the labeled antibody.
 12. The methodof claim 11, wherein the protein-producing cell is an isolatedprotein-producing cell.
 13. The method of claim 11, wherein theprotein-producing cell is one of multiple protein-producing cells in asample
 14. The method of claim 11, wherein the first portion of thecross-linking reagent comprises a F_(AB) fragment configured to bind tothe cell surface marker.
 15. The method of claim 11, wherein the firstportion of the cross-linking reagent comprises a second ligandconfigured to bind to the cell surface marker, and wherein the secondligand does not comprise a F_(AB) fragment.
 16. The method of claim 11,wherein the first ligand is a protein-specific antibody thatspecifically binds to the protein of interest.
 17. The method of claim11, wherein exposing the first ligand is bound to a substrate.
 18. Themethod of claim 17, wherein the first ligand is bound to the substratevia a biotin/streptavidin linkage
 19. The method of claim 17, whereinthe substrate is a bead, a cell, or a combination thereof.
 20. Themethod of claim 11, wherein the protein of interest is an antibody. 21.The method of claim 20, wherein the labeled antibody is an anti-IgGantibody.